Denaturation fingerprinting: two related mutation detection methods especially advantageous for high G + C regions.
Two versions of denaturation fingerprinting (dnF2R and dnF1R are described for detecting mutations. DnF2R is a sensitive screening method in which fingerprints are generated by performing denaturing gel electrophoresis on bidirectional "cycle-sequencing" reactions with each of two dideoxy terminators, e.g. ddATP and ddCTP. When the fingerprints generated by ddATP and ddCTP are combined, all sequence changes are expected to result in one extra and one absent segment. DnF2R was performed on 246- and 318-bp segments of the human factor IX gene, and the products were electrophoresed through a 6% Long Ranger gel with 7 M urea. All 32 single-base mutations were detected in hemizygous males with hemophilia B. DnF2R has been applied to detect a total of seven heterozygous sequence changes in large-scale screening and was found to be especially suitable for high G+C regions. In a blinded analysis, all of twenty-four additional single-base mutations were detected, but 7 of 31 heterozygous mutations were missed (23%). To reduce the effort of dnF2R by almost twofold while retaining the ability to detect all types of single-base changes, one fingerprint (dnF1R) was generated by performing a single reaction with ddATP and a second chemically modified terminator (e.g., ROX-conjugated ddCTP), which retards the mobility of the same termination products. The sensitivity and specificity of dnF1R equaled that of dnF2R, with the exception that the blinded analysis of heterozygotes in the 318-bp segment, which revealed the presence of an additional mutation. DnF under partially denaturing conditions may have optimal sensitivity for the detection of heterozygotes.
Liu
Q
Q
City of Hope National Medical Center, Duarte, CA, USA.
Weinshenker
B G
BG
Wingerchuk
D M
DM
Sommer
S S
SS
D016172:DNA Fingerprinting / Q000379:methods*; D005164:Factor IX / Q000235:genetics; D006801:Humans; D009154:Mutation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D014409:Tumor Necrosis Factor-alpha; D005164:Factor IX